Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, resulting in 1 to 2 million deaths annually. Serious infection and death result primarily from the ability of S. pneumoniae to cause pneumonia in the elderly, young children, and those with underlying conditions. Essential to the ability to cause disease are the capsular polysaccharides. Although 91 distinct capsular serotypes have been identified, all perform similar functions. In systemic infections, high levels of capsule production are necessary for resistance to complement-mediated opsonophagocytosis. In contrast, low levels of capsule are sufficient to sustain colonization in the nasopharynx, as factors necessary for adherence are unmasked as capsule is reduced. The ability to alter capsule production is critical to the survival of S. pneumoniae. For 89 of the 91 S. pneumoniae capsular serotypes, the polymer is assembled by the Wzy-dependent mechanism, which is also used in the assembly of most capsules and exopolysaccharides produced by Gram-positive bacteria, and many capsules and lipopolysaccharide O-antigens of Gram-negative bacteria. Although a general picture for Wzy-dependent synthesis in Gram-positive bacteria has emerged, much remains to be learned about specific aspects of this process. As a model system, we have used the S. pneumoniae serotype 2 capsule, in which the repeat unit contains a backbone of glucose-rhamnose-rhamnose-rhamnose and a side chain of glucose- glucuronic acid. During polymer assembly, repeat units synthesized on the inner face of the cytoplasmic membrane are transported to the outer face of the membrane and then polymerized by the Wzy-polymerase into long chain polymer. In S. pneumoniae, the polymer remains largely cell-associated via linkages to the membrane and peptidoglycan. Modulation of capsule chain length and amount occurs, at least in part, through the action of a phosphoregulatory system. This research proposal focuses on further defining the complex mechanisms involved in Wzy-dependent capsule assembly and control in S. pneumoniae. The specific aims for this proposal are to: 1) characterize the functions of the initiating glycosyltransferase Cps2E and the effects of cps2E mutations; 2) identify glycosyltransferases catalyzing type 2 repeat unit synthesis; 3) characterize mechanisms involved in retention of the capsule on the bacterial cell; and 4) characterize protein-protein interactions between capsule-related proteins. The results of these studies could have broad relevance for understanding mechanisms involved in synthesis and modulation of similar polymers produced by other pathogenic bacteria. Streptococcus pneumoniae is responsible for more than 50,000 deaths annually in the United States and 1 to 2 million worldwide due primarily to its ability to cause pneumonia in the elderly and young children. The proposed studies will further our understanding of how the bacterium synthesizes its major virulence determinant, the capsular polysaccharide. By understanding the mechanisms involved in this process, we may be better able to develop means for treating and preventing these infections.